The development of a reverse genetics system for influenza viruses allows us to site-specifically mutate the genomes of these negative strand RNA viruses. Thus, we are now able to perform structure-function studies on proteins in the context of infectious virus and to define specific virulence characteristics on a molecular level. Using mutational analysis, we will study the functional and antigenic determinants of the influenza virus neuraminidase (NA) in the cytoplasmic tail, the stalk and the head region. The characterization of genetically manipulated influenza viruses (transfectants) will define in a more precise way how the NA influences host range or neurovirulence. Similar studies will be done on the hemagglutinin (HA) in order to define the role of palmitoylation of the HA in the formation of infectious particles and to study host range and virulence as it is influenced by the HA. In order to facilitate the rapid isolation of transfectants with changes in non- HA/non-NA genes, attempts will be made to develop convenient systems which will allow for the rapid exchange of any influenza virus gene with a cDNA-derived RNA segment. We will also develop protocols to establish a reverse genetics system for influenza B viruses.